Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection

Abstract

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2- a]pyridine-3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 μ1) on to a precolumn filled with C 18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C 8 type and the mobile phase was acetonitrile–methanol–phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min −1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml −1, respectively, in human plasma.